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Objective:Artemisia annua L. (Asteraceae) (artemisinin) has special antitumor activity against melanoma, breast, ovarian, prostate, central nervous system and renal cancer cell lines. The present study was conducted to compare effects of free and niosomal formulation of artemisinin on Chronic Myelogenous Leukemia (CML) using K562 cell line. Materials and Methods: Different concentrations of artemisinin were prepared using spectrophotometer in 195 nm wavelength. Two different methods of reverse phase evaporation and ether injection were adopted to prepare the niosomes formulations. Encapsulation, in vitro release, in vitro cytotoxicity determination and the K562 cells viability rates were assessed. Results: The percentage of encapsulation for niosome formulation obtained with reverse phase evaporation and ether injection methods were 87.27±0.93 and 74.9±4.2, respectively. The rate of the artemisinin release across the dialysis membrane was significantly slower for artemisinin-loaded niosomes than that of the free artemisinin (p<0.05). The percentage of K562 cells apoptosis in free artemisinin was significantly higher than that of the niosomal formulations (p<0.05). The niosomal formulations showed significantly more influence than that of the free artemisinin (p<0.05). Conclusion: The use of niosomal formulation of artemisinin could effectively improve its therapeutic index, release rate and apoptosis influence.